Not applicable
The present invention relates to an improvement to recombinant live vaccines integrating and expressing in vivo one or more heterologous genes. It relates in particular to such adjuvant-containing vaccines, to the use of particular adjuvant compounds for using such vaccines as well as to vaccination methods relating thereto. Its subject is also a method of preparing these vaccines.
It is conventional to incorporate into inactivated or subunit vaccines adjuvants intended to increase the immune response towards the antigens which these vaccines contain.
It has also been found to incorporate adjuvants into attenuated live vaccines when the attenuation of microorganisms leads to a reduction in the immune response.
Recently, combined vaccinations against several pathogens using an inactivated vaccine for one valency and an attenuated live vaccine for the other valency have also been proposed. It has thus been proposed to reconstitute the attenuated live vaccine, preserved in freeze-dried form, in the composition of an adjuvant-containing inactivated vaccine. The said composition acts as reconstitution vehicle for the live vaccine, without any adjuvant effect being sought for it.
EP-A-532 833 proposes a vaccine against horse rhinopneumonia, a pathology caused by the equine herpesvirus (EHV). The vaccine is an inactivated and adjuvant-containing vaccine, grouping together the inactivated EHV-4 virus and EHV-1 virus, containing the adjuvant Havlogen(copyright), based on a polyacrylic polymer.
As for most herpesviruses, there is currently no effective vaccine allowing rapid elimination of the virus after infection. The known vaccines attempt to protect against the appearance of clinical signs. In general, the effect on viral excretion remains limited.
According to EP-A-532 833, the vaccine developed is thought to lead to a drop in viral excretion ranging from 79 to 93% (see results section). Eight control animals out of nine excreted virus after a challenge over an average of 1.4 days whereas the normal duration of excretion after challenge is usually greater than or equal to 5 days. This represents a challenge of low intensity which artificially increases the protection of vaccinated horses compared with the controls. The reduction in viral excretion is not therefore significant in this experiment.
Adjuvants of the carbomer type have also been used in inactivated virus-containing equine influenza vaccines (IEV).
Mumford et al (Epidemiol. Infect. (1994), 112, 421-437) recall that two equine IEV vaccine doses are required to induce a transient humoral response and a weak protection against the virus in horses. The authors compare the adjuvant effects of carbomer and of aluminium phosphate on inactivated vaccines in the presence or otherwise of tetanus toxoid. In all cases, a low antibody titre measured by the SRH (single radial haemolysis) technique with respect to the strains H7N7 and H3N8 is obtained after a first vaccination and a second and then third vaccination are necessary to see the appearance of stronger transient responses.
U.S. Pat. No. 4,500,513 also presents vaccination trials against the equine influenza virus with an inactivated vaccine in the presence of a carbomer. The origin of the animals and their medical status is not indicated precisely and it appears that they are ground animals (column 11, 2nd paragraph). The high antibody titres, measured by a haemagglutination inhibition technique, indicate that the animals had probably already been infected with influenza and that the response induced after vaccination was of the booster type, and not of the primary vaccination type.
Finally, Fort Dodge Solvay markets inactivated equine influenza vaccines (Duvaxyn(copyright) IE and IE-T plus) and an inactivated equine rhinopneumonia vaccine (Duvaxyn(copyright) EHV1,4), in a carbomer adjuvant.
Commercial inactivated vaccines against equine influenza, containing the adjuvant aluminium hydroxide (for example Tetagripiffa(copyright), Mxc3xa9rial, Lyons, France) are also known.
A large number of other adjuvants are used in the context of conventional inactivated or subunit vaccines. There may be mentioned, for example, aluminium hydroxide, aluminium phosphate, Avridine(copyright), DDA, monophosphoryl lipid A, Pluronic L121 and other block polymers, muramyl peptides, saponins, trehalose dimycolate, copolymers of maleic anhydride and ethylene, copolymers of styrene and acrylic or methacrylic acid, polyphosphazene, oily emulsions and the like.
WO-A-94 16681 suggests supplementing a recombinant live vaccine expressing a heterologous gene of an enveloped virus with an adjuvant vaccine composition in the form of a water-in-oil, oil-in-water or water-in-oil-in-water emulsion.
Such a solution may however have a number of disadvantages.
In practice, the final user should have available, on the one hand, a freeze-dried active ingredient and, on the other hand, an already constituted emulsion which should make it possible to reconstitute the freeze-dried active ingredient.
Lack of stability of the emulsion during storage could be detrimental to the efficacy and safety of the reconstituted vaccine.
The activity of attenuated live microorganisms could be called into question following their instability in the oily phase. This may in particular be the case for viruses which may thereby lose their viability.
Vaccines in emulsion can also pose problems of safety at the site of injection.
The present invention is therefore given with the objective of providing new vaccine compositions based on recombinant live vaccine expressing at least one heterologous nucleotide sequence, especially a heterologous gene, containing an adjuvant which is capable of remarkably increasing the immunity conferred relative to the same vaccine with no adjuvant and which is perfectly suitable for this type of vaccine.
The Applicant has found the carbomer class of compounds were capable of acting as adjuvant under the required conditions for this type of vaccine and this in unexpected proportions. Trials carried out on animal herpesviruses (EHV-1, Equine Herpesvirus) have shown that the supply of carbomer could reduce viral excretion during an experimental infection, in unexpected proportions. Other trials carried out on the equine influenza A virus have made it possible to obtain, surprisingly, in horses, early and very high serological titres, better than those obtained with the best commercial vaccines.
The subject of the present invention is therefore a recombinant live vaccine comprising a viral vector incorporating and expressing in vivo a heterologous nucleotide sequence, preferably a gene for a pathogenic agent, and at least one adjuvant compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative.